US 8193333 B2
The invention relates to a double-stranded short interfering nucleic acid (siNA) molecule specific to the Bcl-XL transcript, comprising a sense and an antisense region, wherein the sense region comprises the nucleotide sequence SEQ ID NO: 1 or a sequence having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity with said sequence, and the antisense region comprises a nucleotide sequence that is complementary to the sense region, and its use for treating cancer.
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a double-stranded short interfering nucleic acid molecule specific to the Bcl-XL transcript comprising a sense and an antisense region, wherein the sense region comprises the nucleotide sequence SEQ ID NO: 1 or a sequence having at least 70% identity with said sequence, and the antisense region comprises a nucleotide sequence that is complementary to the sense region, and wherein each strand comprises 15 to about 30 nucleotides, and each strand comprises 15 to about 30 nucleotides that are complementary to the nucleotides of the other strand, and
a short interfering nucleic acid molecule targeting the Mcl-1 transcript consisting of a sense strand of the sequence SEQ ID NO: 7 and an antisense strand of the sequence SEQ ID NO: 8.
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This application is a national stage application filed under 35 U.S.C. 371 of International Application No. PCT/IB2007/002762, filed Jun. 22, 2007, which claims priority from International Application No. PCT/IB2006/002685, filed Jun. 26, 2006.
The invention relates to short nucleic acid molecules specific to the the Bcl-XL mRNA that down-regulate the expression of the Bcl-XL protein by RNA interference and inhibit the growth of tumor cells in vivo, and to their use for cancer therapy.
Apoptosis is regulated in part by the Bcl-2 protein family, including pro-apoptotic Bax, Bcl-XS, Bak, Bad, Bid, Bik, Bim, and anti-apoptotic Bcl-2, Bcl-XL, Mcl-1, A1 and Bcl-W. The susceptibility of cells to apoptosis induced by various stimuli such as cytotoxic drugs, serum starvation and radiations appears to be determined, at least in part, by the subcellular localization and the relative ratio between pro- and anti-apoptotic proteins, which can heterodimerize and titrate one another's function (White, E., Genes Dev., 1996, 10, 1-15; Korsmeyer, S. J., Cancer Res., 1999, 59, 1693s-1700s; Cory, S. and Adams, J. M., Nat. Rev. Cancer, 2002, 2, 647-656).
High expression levels of anti-apoptotic members of Bcl-2 family have been found in many tumors, and up regulation of Bcl-2 and Bcl-XL has been shown to be a key element in malignancy and drug resistance (Reed, J. C., Semin. Hematol., 1997, 34, 9-19; Konopleva et al., Br. J. Haematol., 2002, 118, 521-534; Lebedeva et al., Cancer Res., 2000, 60, 6052-6060; Liu et al., Gynecol. Oncol., 1998, 70, 398-403; Simonian et al., Blood, 1997, 90, 1208-1206). In particular, Bcl-2 and/or Bcl-XL are frequently overexpressed in ovarian, nasopharyngeal, breast, prostate and colon carcinoma, glioma, mesothelioma, and melanoma (He et al., Chin. J. Cancer, 2003, 22, 11-15; Min et al, Chin. J. Oncol., 2004, 26, 14-16; Krishna et al., J. Neurosurg., 1995, 83, 1017-1022; Kajewski et al., Am. J. Pathol., 1997, 150, 805-814; Cao et al., Am. J. Respir. Cell. Biol., 2001, 25, 562-568; Soini et al., Clin. Cancer Res., 1999, 5, 3508-3515; Kojima et al., J. Biol. Chem., 1998, 273, 16647-16650; Olopade et al., Cancer J. Sci. Am., 1997, 3, 230-237; Zapata et al, Breast Cancer Res. Treat., 1998, 47, 129-140; Simonian et al., Blood, 1997, 90, 1208-1216; Leiter et al., Arch. Dermatol. Res., 2000, 292, 225-232; Nicholson et al., Nature, 2000, 407, 810-816; Krajewska et al., Cancer Res., 1996, 56, 2422-2427; Maurer et al., Dig. Dis. Sci., 1998, 43, 2641-2648; Mercatante et al., J. Biol. Chem., 2002, 277, 49374-49382; Ferrandina et al., Cancer Lett., 2000, 155, 19-27; Liu et al., Gynecol. Oncol., 1998, 70, 498-503; Marone et al, Clin. Cancer Res., 1998, 4, 517-524; French Patent Application FR 0108864). However, Bcl-XL is generally considered more efficient than Bcl-2 to suppress apoptosis induced by cytotoxic drugs and radiations (Simonian et al, precited; Gottschalk et al., P.N.A.S., 1994, 91, 7350-7354). Therefore Bcl-XL represents a good target for cancer therapy, especially for cancers resistant to conventional anticancer agents.
Transcription of the BCL2L1 gene (BCL2-like 1, BCL2L, BCLX, Bcl-X, bcl-x, or BCL-X gene) produces alternatively spliced variants encoding three isoforms: the longer anti-apoptotic isoforn (Bcl-XL), the shorter pro-apoptotic isoform (Bcl-XS) and a Bcl-X (beta isoform).
Bcl-XL is a 233 amino acids protein encoded by the longer transcript variant (transcript variant 1) comprising Exon 1, Exon 2 and Exon 3 of the BCL2L1 gene: the human Bcl-XL mRNA and protein correspond to NCBI accession numbers NP—612815 (SEQ ID NO: 3) and NM—138578 (SEQ ID NO: 2), respectively.
Bcl-XS is a 170 amino acids protein having the N-terminal sequence (positions 1 to 125) and the C-terminal sequence (positions 189 to 233) of the Bcl-XL protein but lacking a 63 amino acids sequence from positions 126 to 188 of the Bcl-XL protein. Bcl-XS is encoded by the shorter transcript variant (transcript variant 2) comprising Exon 1, the 5′ sequence of Exon 2 and Exon 3. Bcl-XS mRNA lacks the 189 nucleotides sequence located at the 3′ end of Exon 2, that is specific to the Bcl-XL transcript. Also, the corresponding 63 amino acids sequence of the Bcl-XL protein, which is missing in Bcl-XS, is specific to Bcl-XL protein. The human Bcl-XS mRNA and protein correspond to NCBI accession numbers NP—001182 and NM—001191, respectively.
Bcl-X (beta) is a 227 amino acids protein having the N-terminal sequence of Bcl-XL (positions 1 to 188 of Bcl-XL protein, encoded by Exon 1 and Exon 2 of the BCL2 gene), and a C-terminal sequence of 39 amino acids, encoded by Intron 2 of the BCL2 gene.
The Bcl-XL is located at the outer mitochondrial membrane and appears to regulate cell death by blocking the releases of the caspase activator and cytochrome c, from the mitochondrial membrane (Desagher S. and Martinou, J. C., Trends Cell. Biol., 2000, 10, 369-377; Hengartner, M. O., Nature, 2000, 407, 770-776; Kroemer, G., Nat. Med., 1997, 3, 614-620 ; Kroemer et al., Immunol. Today, 1997, 18, 44-51; Marchetti et al., J. Exp. Med., 1996, 184, 1155-1160; Minn et al., Nature, 1997, 385, 353-357; Susin et al., J. Exp. Med., 1996, 184, 1331-1341).
Down-regulation of Bcl-XL expression by antisense oligonucleotides was shown to induce apoptosis and to potentiate the cytotoxic effect of chemotherapy on cancer cells (US Patent Application US 2003/0191300; U.S. Pat. Nos. 5,776,905 and 6,143,291; International PCT Applications WO 00/01393, WO 00/66724; Lebedeva et al., precited ; Yang et al., The J. Biochem., 2003, 278, 25872-25878; Sonnemann et al., Cncer Letters, 2004, 209, 177-185; Sonnemann et al., Int. J. Oncol., 2004, 25, 1171-1181; Ozvaran et al., Mol. Cancer Therapeutics, 2004, 3, 545-550; Smythe et al., The J. Thoracic and Cardiovascular Surgery, 2002, 123, 1191-1198; Simoes et al., Int. J. Cancer, 2000, 87, 582-590, Hopkins-Donaldson et al., Int. J. Cancer, 2003, 106, 160-166; Heeres et al., Int. J. Cancer, 2002, 99, 29-34; Taylor et al., Oncogene, 1999, 18, 4495-4504; Wacheck et al., British Journal of Cancer, 2003, 89, 1352-1357; Taylor et al., Nature Biotechnology, 1999, 17, 1097-1100; Mercatante et al., precited; Frankel et al., Cancer Research, 2001, 61, 4837-4841; Roy et al., Oncogene, 2000, 19, 141-150). However, antisense oligonucleotide technology faces many problems including low absorption rates, non-specific inhibition effects, large effective dosage and toxicity.
The successful use of small interfering RNAs (siRNAs) technology for inhibiting the expression of a specific target holds great promise for the development of new treatment for cancer.
RNAi interference is the process where the introduction of double-stranded RNA into a cell inhibits gene expression in a sequence dependent fashion (reviewed in Shuey et al., Drug Discovery Today, 2002, 7, 1040-1046). RNAi has been observed in a number of organisms such as mammalian, Drosophila, nematodes, fungi and plants and is believed to be involved in anti-viral defense, modulation of transposon-activity and regulation of gene expression. RNAi is usually described as a post-transcriptional gene-silencing mechanism in which dsRNA triggers degradation of homologous messenger RNA in the cytoplasm. Target recognition is highly sequence specific since one or two base pair mismatches between the siRNA and the target gene will greatly reduce silencing effect. The mediators of RNA interference are 21-and 23-nucleotide small interfering RNAs (siRNA). In a second step, siRNAs bind to a ribonuclease complex called RNA-induced silencing complex (RISC) that guides the small dsRNA to its homologous mRNA target. Consequently, RISC cuts the mRNA approximately in the middle of the region paired with the antisens diRNA, after which the mRNA is further degraded.
Administration of siRNA to mice was shown to inhibit efficiently the expression of a target located in various organs (liver, brain, eyes, lung, kidney) as well as in xenografts (Braasch et al., Biochemistry, 2003, 42, 7967-7975; Duxbury et al., Biochem. Biophys., Res. Comm., 2003, 311, 786-792; Giladi et al., Mol. Ther., 2003, 8, 769-776; Lewis et al., Nat. Genet., 2002, 32, 107-108; Makimura et al, BMC Neurosci., 2002, 3, 18-; Reich et al., Mol. Vis., 2003, 9, 210-216; Song et al., Nat. Med., 2003, 9, 347-351; Zender et a., P.N.A.S., 2003, 100, 7797-7802.
Compared with antisense technology, RNAi is an efficient gene-specific technology, and has advantages of long-term stability, reversibility, and simple procedures.
RNAis targeting Bcl-XL have been used, in vitro to study the role of Bcl-XL in cell survival and resistance to chemotherapy. Down-regulation of Bcl-XL expression by RNAi was shown to induce apoptosis and to potentiate the cytotoxic effect of chemotherapy on cancer cells, in vitro (Taniai et al., Cancer Res., 2004, 64, 3517-3524; Zhang et al., Haematologica, 2004, 89, 1199-1206; Zender et al., Hepatology, 2005, 41, 280-288; Shimizu et al., Nature Cell Biology, 2004, 6, 1221-1228; Hon et al., J. Immunol., 2004, 173, 4425-4432; Dodier et al, Gynecologic Oncology, Epub, Sep. 26, 2005; He et al., Chinese J. Cancer, 2005, 24, 646-652; Liu et al., Acta Pharmacol., Sin., 2005, 26, 228-234; Lei et al., Acta Biochimica et Biophysica Sinica, 2005, 37, 555-560; Pizzi et al., Cell Death and Differenciation, 2005, 12, 761-772; Rorhbach et aL., J. Mol. Cell. Cardio., 2005, 38, 485-493; Tran et al., The J. Biol., Chem., 2005, 280, 3483-3492; Zhu et al., Cancer Biology and Therapy, 2005, 4, 393-397; Zhu et al., Molecular Cancer Therapeutics, 2005, 4, 451-456; US Patent Application 2005/0176025).
However, few RNAis are specific to Bcl-XL (Zender et al.; Hon et al.; Dodier et al.; He et al., Liu et al., Tran et al., Zhu et al., Cancer Biology and Therapy, precited). Furthermore, some of the Bcl-XL-specific siRNAis have been tested only in combination with non-specific siRNAis (Dodier et al. precited) and none of the Bcl-XL-specific siRNAis was proven to have an antitumoral effect in vivo.
The inventors have engineered Bcl-XL-specific siRNA able to down-regulate efficiently the expression of the anti-apoptotic protein Bcl-XL. Injection of low dose of the Bcl-XL-specific siRNA to mice implanted with human chemoresistant tumor cells increases considerably the mice survival and induces a complete regression of the pre-established tumor for at least 5 months after tumor cells implantation.
This siRNA is useful in cancer therapy, in particular for treating tumors which are resistant to conventional anticancer agents.
Therefore, the invention relates to a double-stranded short interfering nucleic acid (siNA) molecule specific to the Bcl-XL transcript, comprising a sense and an antisense region, wherein the sense region comprises the nucleotide sequence SEQ ID NO: 1 or a sequence having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity with said sequence, and the antisense region comprises a nucleotide sequence that is complementary to the sense region.
The short interfering nucleic acid (siNA) molecule according to the invention is specific to the Bcl-XL transcript (mRNA) and targets 19 contiguous nucleotides corresponding to positions 849 to 867, by reference to the human sequence NCBI accession number NM—138578 (SEQ ID NO: 2 in the attached sequence listing). The target of the siNA molecule according to the invention is included in the 189 nucleotides sequence at the 3′ end of Exon 2 of the BCL2L1 gene (positions 742 to 930 of SEQ ID NO: 2). This target is absent in Bcl-XS transcript. Furthermore, the target is located in a region of the Bcl-XL transcript which is outside the regions of homology with the Bcl-2 transcript (positions 741 to 843 and 907 to 947 of SEQ ID NO: 2).
The siNA molecule according to the present invention inhibits specifically the Bcl-XL mRNA and protein expression. The siNA has no effect on the expression of the pro-apoptotic Bcl-XS protein or other Bcl-2 family members such as Bcl-2 and Mcl-1.
Inhibition of Bcl-XL expression may be assessed by any RNA or protein analysis technique, which is well-known in the art (Northem-blot, Western-blot, quantitative RT-PCR).
In addition the siNA molecule of the present invention has an antitumoral activity in vivo, that can be assayed in appropriate animal models known to those of ordinary skill in the art.
The invention encompasses the synthetic, semi-synthetic or recombinant siNA that inhibit the expression of the Bcl-XL mRNA and protein from any organism. SEQ ID NO: 1 is the human target sequence, i.e., the portion of the human mRNA which is complementary to the antisense region of the siNA molecule. Given the positions of the target in the human mRNA sequence, one skilled in the art will easily find the corresponding positions in the homologous sequences of other organisms (eukaryotes, for example mammals) which are accessible in the data bases such as the NCBI database (htt://www.ncbi.nlm.nih.gov/). Such homologous sequences can be identified as is known in the art, for example using sequence alignment. In addition, the siNA molecule of the invention may inhibit the expression of target gene variants, for example polymorphic variants resulting from haplotype polymorphism.
siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions and quadruple base interactions, that can provide additional target sequences. For example, the siNA molecule can be designed to target a sequence that is unique to a specific target gene mRNA sequence (a single allele or single nucleotide polymorphism (SNP)) due to the high degree of specificity that the siNA molecule requires, to mediate RNA activity. Alternatively, when mismatches are identified, non-canonical base-pairs (for example mismatches and/or wobble bases) can be used to generate siNA molecule that target more than one sequence. In a non-limiting example, non-canonical base-pairs such as uu and cc base pairs are used to generate siNA molecules that are capable of targeting homologous target mRNA sequences. In this approach, a single siNA can be used to inhibit expression of more than one mRNA instead of using more than one siNA molecule to target the different mRNAs.
In one embodiment, the invention features a siNA molecule wherein each strand comprises or consists of 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and each strand comprises at least 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) nucleotides that are complementary to the nucleotides of the other strand. For example, the siNA molecule of the invention comprises or consists of a 19 to 21-nucleotide duplex (19 to 21 base pairs).
In another embodiment of the invention, the siNA molecule comprises or consists of ribonucleotide(s) (2′-OH nucleotides).
In another embodiment, the invention features a siNA molecule wherein the sense region comprises or consists of the sequence SEQ ID NO: 1 and the antisense region comprises or consists of the sequence SEQ ID NO: 4. This siNA targets the human gene (Table I).
In another embodiment of the invention, the siNA molecule compnses overhanging nucleotide(s) at one or both end(s), preferably, 1 to about 3 (e.g. about 1, 2, or 3) overhanging nucleotides. The overhanging nucleotides which are advantageously at the 3′ end of each strand, are preferably 2′-deoxynucleotide(s), preferably 2′deoxypyrimidine(s), such as a 2′-deoxythymidine(s). For example, the siNA molecule of the invention is a 21-nucleotide duplex, with 19 base pairs and 3′-terminal tt overhang(s). In particular, the sense strand is of the sequence SEQ ID NO: 5 and the antisense strand is of the sequence SEQ ID NO: 6; this siNA, noted siXL1, corresponds to SEQ ID NO: 1 (sense strand) and SEQ ID NO: 4 (antisense strand) with tt overhangs added at the 3′ ends.
In another embodiment of the invention, the siNA molecule comprises blunt end(s), where both ends are blunt, or alternatively, where one of the ends is blunt. For example, the siNA molecule of the invention is a 19 to 21-nucleotide duplex, with 19 to 21 base pairs and blunt ends.
In another embodiment of the invention, the siNA molecule is assembled from two separate oligonucleotide fragments or strands, wherein one fragment (sense strand) comprises the sense region and the second fragment (antisense strand) comprises the antisense region of the siNA molecule.
In another embodiment, the invention features a siNA molecule wherein the sense region is connected to the antisense region via a linker molecule, such as a nucleotide or non-nucleotide linker. A nucleotide linker can be a linker of at least 2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Examples of such siNA molecules include small hairpin nucleic acid (shNA) molecules.
A non-nucleotide linker comprises abasic nucleotides, aptamers, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds.
In another embodiment of the invention, the siNA molecule comprises mismatches, bulges, loops or wobble base pairs to modulate the activity of the siNA molecule to mediate RNA interference.
In addition, the siNA molecule may include one or more modifications that increase resistance to nuclease degradation in vivo and/or improve cellular uptake. The siNA may include nucleotides that are modified at the sugar, phosphate, and/or base moiety, and/or modifications of the 5′ or 3′ end(s), or the internucleotidic linkage.
In another embodiment of the invention, the siNA molecule comprises one or more modified pyrimidine and/or purine nucleotides, preferably on each strand of the double-stranded siNA. More preferably, said modified nucleotides are selected from the group consisting of: 2′-O-methylnucleotides, 2′-O-methoxyethylnucleotides, deoxynucleotides, such as 2′-deoxynucleotides and 2′-deoxy-2′-fluoronucleotides, universal base nucleotides, acyclic nucleotides and 5-C-methyl nucleotides. A siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA molecule. The percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand or both the sense and the antisense strands.
In another embodiment, the invention features a siNA molecule wherein the strand comprising the sense region (sense strand) includes a terminal cap moiety at the 5′-end, the 3′-end, or both the 5′ and 3′ ends of the strand, preferably a deoxy abasic moiety or glyceryl moiety.
In another embodiment, the invention features a siNA molecule wherein the strand comprising said antisense region (antisense strand) includes a phosphate group at the 5′-end.
In another embodiment of the invention, the siNA molecule comprises at least one modified internucleotidic linkage, such as a phosphorothioate linkage.
The siNA molecules according to the invention may be produced by chemical synthesis by using well-known oligonucleotides synthesis methods which make use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end and phosphoramidites, at the 3′ end. The nucleic acid molecules of the present invention can be modified to enhance stability by modification with nuclease resistant groups, for example 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, TIBS, 1992, 17, 34 and Usman et al., Nucleic Acids Symp. Ser., 1994, 31, 163). Examples of such modified oligonucleotides include with no limitation: 2′ F-CTP, 2′ F-UTP, 2′ NH2-CTP, 2′ NH2-UTP, 2′ N3-CTP, 2-thio CTP, 2-thio UTP, 4-thio UTP, 5-iodo CTP, 5-iodo UTP, 5-bromo UTP, 2-chloro ATP, Adenosine 5′-(1-thiotriphosphate), Cytidine 5′-(1-thiotriphosphate), Guanosine-5′-(1-thiotriphosphate), Uridine-5′-(1-thiotriphosphate), Pseudo-UTP, 5-(3-aminoallyl)-UTP and 5-(3-aminoallyl)-dUTP. siNA contructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC) and re-suspended in water.
The chemically-synthesized siNA molecule according to the invention may be assembled from two distinct oligonucleotides which are synthesized separately. Alternatively, both strands of the siNA molecule may be synthesized in tandem using a cleavable linker, for example a succinyl-based linker.
Alternatively, the siNA molecules of the invention may be expressed (in vitro or in vivo) from transcription units inserted into DNA or RNA vectors known to those skilled in the art and commercially available.
The invention relates also to a transcription unit comprising: a transcription initiation region, a transcription termination region, and a nucleic acid sequence encoding a least one siNA molecule according to the present invention, wherein said nucleic acid sequence is operatively linked to said initiation region in a manner that allows expression and/or delivery of the siNA molecule.
The nucleic acid sequence may encode one or both strands of the siNA molecule, or a single self-complementary strand that self-hybridizes into a siNA duplex.
The transcription initiation region may be from a promoter for a eukaryotic RNA polymerase I, II or III (pol I, II or III). Transcripts from pol II or pol II promoters are expressed at high levels in all cells. Alternatively, prokaryotic RNA polymerase promoters may be used, providing that prokaryotic RNA polymerase enzyme is expressed in the appropriate cells. Transcription units derived from genes encoding U6 small nuclear transfer RNA and adenovirus VA RNA are useful in generating high concentrations of desired siNA in cells.
The invention concerns also an expression vector comprising a nucleic acid encoding at least one siNA molecule of the instant invention. The expression vector may encode one or both strands of the siNA molecule, or a single self-complementary strand that self-hybridizes into a siNA duplex. The nucleic acid encoding the siNA molecule of the instant invention is preferably inserted in a transcription unit as defined above.
Large numbers of DNA or RNA vectors suitable for siNA molecule expression are known to those of skill in the art and commercially available. The recombinant vectors can be DNA plasmids or viral vectors. SiNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered in vivo, and persist in target cells. Alternatively, viral vectors can be used to provide transient expression of siNA molecules.
The invention concerns also eukaryotic or prokaryotic cells which are modified by a vector as defined above.
The invention concerns also a pharmaceutical composition comprising at least a siNA molecule or an expression vector, as defined above, in an acceptable carrier, such as a stabilizer, a buffer and the like.
A pharmaceutical composition or formulation refers to a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, inhalation, or by injection. These compositions or formulations are prepared according to any method known in the art for the manufacture of pharmaceutical compositions.
In one embodiment, the invention features a composition wherein the siNA molecule or vector is associated to a compound that allows the delivery of the siNA/vector into the cancer cells and/or endothelial cells. The compound may be a membrane peptide, transporter, lipid, cationic polymer, PEI. Preferably, the siNA and the compound are formulated in microspheres, nanoparticules or liposomes. Furthermore, the siNA molecule or vector may be associated with a compound that allows a specific targeting of the tumor and/or endothelial cells, such as a ligand of a cell-surface antigen or receptor, for example a peptide such as a RGD peptide, a sugar, a folate or an antibody specific for said antigen/receptor.
In another embodiment, the invention features a composition comprising a combination of at least two different siNA molecules.
In particular, the composition comprises a siNA molecule as defined above and an other siNA molecule having an antitumoral effect.
siNA molecules having an antitumoral effect include with no limitations siNA targeting the Mcl-1 transcript. The siNAs consisting of a sense strand of the sequence SEQ ID NO: 7, 19 or 20 and an antisense strand of the sequence SEQ ID NO: 8, 21, and 22, respectively, are examples of Mcl-1-specific siRNAs.
In another embodiment, the invention features a composition wherein the siNA molecule or vector is associated with at least one anticancer drug.
The invention also concerns a siNA molecule or a vector as defined above, as a medicament.
The invention concerns also the use of a siNA molecule or a vector as defined above, for the manufacture of a medicament for treating cancer.
The invention concerns further the use of a combination of a short interfering nucleic acid molecule specific to the Bcl-XI transcript as defined hereabove (siNA or an expression vector comprising said siNA) and of a short interfering nucleic acid molecule targeting the Mcl-1 transcript for the manufacture of a cytotoxic medicament for treating cancer.
The cancer may be of any type. Preferably, the cancer is a solid tumor, for example an ovarian, nasopharyngeal, breast, prostate or colon carcinoma, a glioma, a mesothelioma or a melanoma.
In one embodiment of said use, the siNA molecule or vector is associated with an anticancer drug.
The invention concerns also a product containing at least a siNA molecule or vector as defined above, and an anticancer drug, as a combined preparation for simultaneous, separate or sequential use in anticancer therapy.
The anticancer drugs which are used in combination with the siNA molecule or the vector according to the invention are those commonly used in chemotherapy, and include: cytotoxic agents, such as alkylating agents, antimitotic agents and antimetabolites, anti-angiogenic factors, tyrosine kinase inhibitors, and BH3-domain mimetics.
BH3 mimetics are compounds which have a function similar to Bak BH3 peptide and bind to the hydrophobic pocket of the anti-apoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-XL. BH3I-2′ (3-iodo-5-chloro-N-[2-chloro-5-((4-chlorophenyl)sulphonyl)phenyl]-2-hydroxybenzamide), HA14-1 (Ethyl 2-amino-6bromo-4-(1-cyano-2-ethoxy-2ethoxy-2oxoethyl)-4H-chromene-3-carboxylate), YC137 (2-Methoxycarbonylamino-4-methylsulfanyl-butyric acid, 4-(4,9-dioxo-4,9-dihydronaphto[2,3-d]thiazol-2ylamino)-phenylester) and ABT737 are examples of BH3 mimetics.
Preferred anticancer drugs are cisplatin (cis-diaminedichoroplatinum, CDDP or DDP), carboplatin, doxorubicine, pemetrexed (Alimta®), paclitaxel (Taxol®) and BH3 mimetics.
The siNA molecule according to the present invention is generally used as an adjuvant therapy following the surgical resection of the tumor(s). In addition, the siNA molecule according to the invention may be used in combination with other conventional anticancer therapies including radiotherapy and immunotherapy.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence or treat (alleviate a symptom to some extent, preferably all the symptoms) of a disease or state. The pharmaceutically effective dose of the siNA depends upon the type of cancer, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors, that those skilled in the medical arts will recognize. Generally, an amount between 1 μg/kg and 100 mg/kg, preferably between 5 μg/kg and 100 μg/kg, body weight/day of active ingredients is administered.
The siNA of the invention may be administered by a single or multiple route(s) chosen from: intratumoral, for example intracerebral (intrathecal, intraventricular), percutaneous, subcutaneous, intravenous, intramuscular, intraperitoneal, intrarachian, oral, sub-lingual, or inhalation.
When the siNA molecule or vector is used in combination with chemotherapy or radiotherapy, it is preferably administered prior to the anticancer agent, more preferably at least 48 hours prior to the anticancer agent.
In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, which refers to examples illustrating the siNA molecules and their uses according to the invention, as well as to the appended drawings in which:
B. exposure to 20 μg/ml cisplatin. C20: exposure to 20 μg/ml cisplatin alone. siRNA control/C20: exposure to a combination of control siRNA with 20 μg/ml cisplatin. siXL1/C20: exposure to a combination of siXL1 with 20 μg/ml cisplatin
The following examples illustrate the invention but in no way limit it.
1) Material and Methods
a) Cell Lines
SKOV3, OAW42, OAW42-R10, IGROV1 and IGROV1-R10 human ovarian adenocarcinoma cell lines, GL15 glioblastoma cell line, and NCI-H28 mesothelioma cell line were obtained from ECACC or ATCC. OAW42, OAW42-R10 cells were grown in DMEM (GIBCO-BRL) supplemented with 10% foetal calf serum (GIBCO-BRL), 2 mM Glutamax™ (GIBCO-BRL), 1 mM sodium pyruvate (GIBCO-BRL), 20 UI/L human recombinant insulin (LILLY), 33 mM sodium bicarbonate and 20 mM HEPES All the other cell lines were grown in RPMI 1640 medium (GIBCO-BRL) supplemented with 10% foetal calf serum (GIBCO-BRL), 2 mM Glutamax™ (GIBCO-BRL), 33 mM sodium bicarbonate and 20 mM HEPES. Cells were maintained at 37° C. in a 5% CO2 humidified atmosphere, and split twice a week by trypsinization.
Cisplatin (CDDP for cis-diamino-dichloro-platinum II) was obtained in the commercial form of Cisplatin from MERCK. Oligofectamine Reagent was obtained from INVITROGEN. It was supplied in liquid form and stored at 4° C. Linear polyethylenimine (L-PEI) was obtained from POLYPLUS-TRANSFECTION as an aqueous 100 mM solution in endotoxin-free water and stored at −80° C. prior to use.
c) In vitro Exposure to Cisplatin
Exponentially growing cells were exposed to CDDP for 2 hours at 37° C., in serum free medium. After exposure to the drug, the cell layers were rinsed and incubated in complete growth medium.
d) SiRNA Design and Transfection
Specific double-stranded 19-nucleotides RNA sequences homologous to the targeted mRNAs were used to silence Bcl-XL and Mcl-1 expression. The sequence of the double-stranded RNA used to block Bcl-XL expression, noted siXL1, is:
The control siRNA does not bear any homology with any relevant human genes (Duxbury et al., 2003, precited). siXL1 targets selectively the Bcl-XL mRNA, but not the Bcl-XS mRNA. The siRNA Mcl-1 targets selectively the long isoform of Mcl-1 mRNA. SiRNAs were synthesized, HPLC purified and annealed by Eurogentec.
The siRNA duplexes were transfected according to the recommended procedure by using the Oligofectamine Reagent and Opti-MEM medium (INVITROGEN LIFE TECHNOLOGIES). 30-40% confluent cells were transfected in 25 cm2 flasks with different concentrations of siXL1, siRNA Mcl-1 or siRNA control and incubated at 37° C., 5% CO2 for 4 hours. Next, 20% fetal bovin serum was added to reach a final concentration of 10% foetal bovin serum in the 25 cm2 flask.
One μg of total RNA, extracted by RNAeasy kit (QIAGEN), was reverse-transcripted with 200 units Omniscript reverse transcriptase (QIAGEN) in first strand reaction conditions recommended by the manufacturer. The targeted cDNA were amplified using the following pair of primers, Bcl-XL/S forward 5′-ttggacaatggactggttga-3′ (SEQ ID NO: 11) and Bcl-XL/S reverse 5′-gtagagtggatggtcagtg-3′ (SEQ ID NO:12 ; Bargou et al., Int. J. Cancer, 1995, 16, 854-859), and amplified under the following cycling conditions: pre-incubation 94° C. for 5 min then 94° C. for 40 sec, 58° C. for 60 sec and 72° C. for 40 sec for 30 cycles, post-incubation 72° C. for 3 min within a Mastercycleur Gradient (EPPENDORF). The use of these primers allows the migration of Bcl-Xs and Bcl-XL PCR products as two distinct bands of respectively 576 and 765 bp.
f) Real-time RT-PCR
One μg of total RNA, extracted by RNAeasy kit (QIAGEN), was reverse-transcripted with 200 units Omniscript reverse transcriptase (QIAGEN) in first strand reaction conditions recommended by the manufacturer. The Glyceraldehyde-3-phosphate deshydrogenase gene (GAPDH) was used to normalise Bcl-XL expression. The Bcl-XL and GAPDH cDNAs were amplified with the use of primers and Taqman-MGB probes which have been selected with the use of the Primer Express Applications-Based Primer Design Software (PERKIN-ELMER APPLIED BIOSYSTEMS).
For Bcl-XL, following primers were used: 5′-tgcgtggaaagcgtagacaa-3′ (SEQ ID NO:13) and 5′-aggtaagtggccatccaagct-3′ (SEQ ID NO:14), together with a Taqman-MGB probe presenting the following sequence: 5′-FAM-agatgcaggtattggtg-TAMRA-3′ (SEQ ID NO: 15).
For GAPDH, primers with the sequences 5′-gcaccgtcaaggctgagaac-3′ (SEQ ID NO: 16) and 5′-tctcgctcctggaagatggt-3′ (SEQ ID NO: 17) were used together with a Taqman-MGB probe presenting the following sequence 5′-VIC-catcaatggaaatccca-TAMRA-3′ (SEQ ID NO: 18). A Basic Local Alignment Search Tool (BLAST) search of the National Centre for Biotechnology Information (NCBI) database revealed no homology of the primer and probe sequences to any other known human genes. Data are presented relative to an untreated control sample chosen as calibrator.
Bcl-XL mRNA and GAPDH MRNA expression were measured separately by real time quantitative RT-PCR using TaqMan technology (ABI PRISM 7000, PE APPLIED BIOSYSTEMS). For each PCR, a master mix was prepared with 2× reaction buffer (qPCR Mastermix, EUROGENTEC) containing dNTP, Hot Goldstar DNA polymerase, 5 mM MgCl2, UNG and ROX. PCR was carried out with 400 nM of each primers for Bcl-XL, 800 nM of each primers for GAPDH and 100 nM of appropriate probe. 5 μl of each diluted cDNA was added to 20 μl of the PCR master mix. Thermal cycling conditions comprised an initial UNG incubation at 50° C. for 2 min, Hot Goldstar DNA polymerase activation at 95° C. for 10 min, 50 cycles of denaturation at 95° C. for 15 sec, and annealing/extension at 60° C. for 1 min. Each run included the five points of the calibration curve for GAPDH and Bcl-XL, the calibrator sample, the experimental samples, and a non-template control, all in triplicate.
Standard curves were established for Bcl-XL and GAPDH cDNA with five-fold serial dilution of Jurkat cell cDNA, which expresses Bcl-XL. Threshold cycle (CT) was used to determine the quantity (Q) of Bcl-XL and GAPDH mRNA. Bcl-XL relative expression was calculated as follow: Bcl-XL expression=(Q Bcl-XL/Q GAPDH)sample/(Q Bcl-XL/Q GAPDH)calibrator. Results were analysed with SDS 2.0 software from APPLIED BIOSYSTEMS.
g) Western Blotting
Cells were rinsed with ice cold PBS and lysed in 150 mM NaCl, 50 mM Tris-HCl pH 8, 1% Triton X100, 4 mM PMSF, 2 mM Aprotinin, 5 mM EDTA, 10 mM NaF, 10 mM NaPPi, 1 mM Na3VO4 for 30 min on ice. Lysates were clarified by centrifugation at 10000 g for 10 min at 4° C. and protein concentrations were determined using the Bradford assay (BIO-RAD). Equal amounts of total cellular protein (20 μg) were resolved in a Bis-tris-HCL buffered (pH 6.4) 4-12% polyacrylamide gel (NuPAGE® Novex® 4-12% Bis-tris gel, INVITROGEN) for 35 min at 200V and electrophoretically transferred on a PVDF membrane (MILLIPORE) for 75 min at 30V. The membrane was blocked for 1 hour at room temperature in T-TBS (132 mM NaCl, 20 mM Tris-HCl pH 7.6, 0.05% Tween 20) supplemented with 5% non-fat dry milk. The membrane was incubated for 1 hour at room temperature in T-TBS-milk with the following primary antibodies: anti-Bcl-XL/S (1:500, S18 SANTA-CRUZ BIOTECHNOLOGY), anti-PARP (1:1000, CELL-SIGNALING TECHNOLOGY), anti-caspase-3 (1:1000, BD BIOSCIENCES PHARMINGEN) anti-cleaved caspase-3 (1:1000, CELL-SIGNALING TECHNOLOGY), anti-alpha-tubulin (1:3000, SIGMA) and anti-Mcl-1 (1:750, S19 SANTA-CRUZ BIOTECHNOLOGY). After three washes with T-TBS, the membrane was incubated for 1 h at room temperature in T-TBS-milk with the adequate peroxidase conjugated secondary antibody (Anti-rabbit IgG, CELL-SIGNALING TECHNOLOGY, and anti-mouse IgG, AMERSHAM). After 3 washes with T-TBS and one with TBS, the immunoreactivity was detected by enhanced chemiluminescence (ECL kit, AMERSHAM).
h) Morphological Characterization of Apoptotic Cells by Nuclear Staining with Diamidino-2-phenylindol (DAPI)
After treatment, detached cells were collected separately and adherent cells were dissociated by trypsin/EDTA. Adherent and detached cells were then pooled and centrifuged at 1500×g for 5 min before being fixed in 70% ethanol. The cells were then collected on a polylysine-coated glass slide by cytocentrifugation, before a 30 min room temperature incubation in a 1 μg/ml DAPI aqueous solution (BOEHRINGER MANNHEIM). Slides were thereafter extensively washed in distilled water and mounted in Mowiol (CALBIO-CHEM).
i) Flow Cytometry Analysis of DNA Cellular Content
Preparation of Cells.
After treatment, detached cells were collected separately. Adherent cells were then harvested by trypsin/EDTA dissociation. Adherent and detached cells were then pooled and washed in PBS before being fixed in 70% ethanol and stored at −20° C. until analysis. Before flow cytometry analysis, the cells were washed in PBS and incubated for 30 min at room temperature in PBS in order to allow the release of low molecular weight (m.w.) DNA, characteristic of apoptotic cells. After a centrifugation at 4000 g for 10 min, the cell pellets were resuspended and stained with propidium iodide (PI) using the DNA Prep Coulter Reagent Kit (BECKMAN-COULTER) at a final concentration of 106 cells/ml.
Samples were analyzed using an EPICS XL flow cytometer (BECKMAN COULTER) equipped with an argon laser at 15 mW. PI-stained cells were analyzed using a 488 mn excitation. A 620 nm band pass filter was put on the red fluorescence of PI. Computerized gating was applied on the side and forward scatter to exclude very small debris and on pulse width and integral peak of red fluorescence to eliminate aggregates. All samples were analyzed at a flow rate lower than 100 events per second and with a sheath pressure of 30 psi.
EXPO 32 Acquisition Software (Beckman Coulter) was run for data acquisition.
a) siXL1 Effect on Bcl-XL Expression, Cell Growth Rate and Apoptosis Induction.
The effects of siXL1, which targets selectively the Bcl-XL mRNA, but not the Bcl-XS mRNA, was tested in various tumour cell lines. SKOV3 cells treatment with siXL1 greatly reduces the Bcl-XL mRNA level, as demonstrated by RT-PCR analysis (
b) Effect of siXL1/Cisplatin Combination on SKOV3 Cells in vitro.
The exposure to the combination of cisplatin and siXL1 induces a high level of cell mortality after several days, in conditions where cisplatin alone induces only a transient arrest in cell cycle progression (5 and 20 μg/ml). A slight effect is detectable after 24 hours, whereas an abundant cell mortality is observed after 4 days of exposure to the combination of siXL1 with cisplatin; the viable cells representing less than 10% compared to the untreated cells (
Gene expression analysis (
The apoptosis induction, as demonstrated by caspase 3 activation and PARP cleavage detection, is greatly amplified by the combination of siXL1 with cisplatin. After 24 h exposure to cisplatin alone, no sign of apoptosis is detectable, whatever the concentration used. By contrast, in cells exposed to siXL1, caspase 3 and PARP cleavage is detectable, this cleavage is amplified in cells co-treated with 20 μg/ml cisplatin and siXL1. After 96 h exposure to cisplatin, the difference is clearly detectable, caspase 3 activation and PARP cleavage reaching their maximum when cisplatin is combined with siXL1.
These results confirm the morphological observations (
These results show that the chosen siRNA sequence, siXL1, inhibits very efficiently Bcl-XL mRNA and protein expression and increases strongly the cytotoxic activity of cisplatin in an ovarian tumour cell line that is very aggressive and highly resistant to cisplatin. The siXL1 effect is specific, persists 96 hours and the optimal concentration of siXL1 is 150 nM.
c) siMcl-1 Effect on Mcl-1 Expression in SKOV3 Cells in vitro.
SKOV3 cells treatment with siMcl-1 reduces Mcl-1 mRNA expression almost completely, as demonstrated by RT-PCR analysis (
d) Effect of siXL1/siRNA Mcl-1 Combination on SKOV3 Cells in vitro.
The exposure to the combination of siXL1 with a siRNA directed against the Mcl-1 mRNA produces a cytotoxic effect on SKOV3 cells (in the absence of cisplatin exposure) at concentrations where each siRNA produces no cytotoxic effect (50 nM;
Cellular and nuclear morphologies observation, as well as cell cycle analysis (
In the same conditions, SKOV3 cells transfected with siXL1 alone (150 nM each) or in combination with control siRNA (75 nM each) show a moderate apoptosis, as demonstrated by nuclear fragmentation and condensation, and a reduction of cell viability (
By comparison, the combination of siXL1 with siMc1 -1 (75 nM each) induces a massive cell detachment and cell death as demonstrated by the cell morphology, the presence of numerous nuclear features of cell death by apoptosis and of an important pre-G1 peak (
1) Material and Methods
a) Nude Mice
4-week-old female Swiss/nude mice were obtained from CHARLES-RIVER LABORATORIES and maintained in a pathogen-free environment. The mice were fed a standard laboratory diet and tap water ad libitum and kept a 23±1° C. with a 12 h light/dark cycle. Animal experiments in the present study were performed in compliance with the guidelines of the Federation of european laboratory animal science associations.
b) PEI/siRNA Complex Formation and Administration
PEI/siRNA complexes were prepared in 5% glucose solution with a ratio of L-PEI nitrogen to siRNA phosphate of 5:1. Various amounts of siRNA (125, 625 or 2500 ng) and L-PEI were diluted separately, and PEI was then added to the siRNA. The solution was quickly homogenized and left for 15 min at room temperature. It was injected intraperitoneally in mice as a 0.5 ml 5% glucose solution.
c) Intraperitoneal Implantation of the Tumor Cells and siRNA Injection in Mice
6-8 weeks old mice were implanted intraperitoneally with 2×107 ovarian adenocarcinoma SKOV3 cells. As described previously, this model reflects the i.p growth pattern of advanced ovarian cancer (Louis et al, Cancer Gene Therapy, 2006, 13, 367-374).
c1) Analysis of Mice Survival
Twenty one days after tumour implantation, mice were allocated to one of the eight treatment groups (10 mice per group) summarized in Table II:
The same treatment was repeated one month later. Mice were killed and autopsied when considered as moribund. Organs and tumours were formalin-fixed and paraffin-embedded for histological examination. At the end of the experiment, all surviving animals were sacrificed, and histological examination was performed in order to detect residual tumour nodes.
After peritoneal carcinomatosis development, animals were injected intraperitoneally with 5, 25 or 100 μg/kg siXL1, naked or complexed with PEI. Mice were killed 3 days after transfection and each organ (kidney, liver, spleen, pancreas, ovary, peritoneum, diaphragm, lung, heart and skeletal muscle) was washed with saline solution. Parts of each organs were either frozen in liquid nitrogen and stored at −80° C. for RT-PCR and western blot analysis, or formalin-fixed and paraffin-embedded for histological examination and immunohistochemical analysis.
d) Subcutaneous Implantation of the Tumor Cells and siRNA Injection in Mice
SKOV3 cells (2×107/500 μL) were inoculated subcutaneously into the right flank of nude mice, and establishment of palpable tumours was determined. The tumour volume was calculated as v=L×l2×p/6 where L and l represent the larger and the smaller tumour diameter measured twice week with digital caliper. When tumours reached an average volume of ˜100 mm3 (on day 10), three experimental groups (three mice per group) were tested as follow: (a) untreated, (b) siXL1 naked (25 μg/kg), and (c) siXL1-PEI (25 μg/kg). The samples were diluted in 0.5 ml 0.9% NaCl solution (siRNA naked) or in 0.5 ml 5% glucose solution (siRNA-PEI) and injected intraperitoneally. This process was repeated once a week.
Immunohistochemical staining was performed on paraffin-embedded material. To perform immunostaining, 4 μm-thick sections were dewaxed, rehydrated and treated 30 minutes by high-temperature-heating antigen retrieval technique in citrate buffer 0.1M pH6 to unmask epitopes. Sections were incubated 1 hour at room temperature with polyclonal antibody anti-Bcl-XL/S (S18) obtained from Santa Cruz (TEBU-BIO) and diluted 1:50. After washes, slides were incubated with Rabbit IgG Vectastain ABC Kit (VECTOR, ABCYS) according to the manufacturer instructions. Staining was revealed with DAB chromogen system (DAKO) and sections were counterstained with hematoxylin.
The effect of siXL1 in vivo was tested in a mouse model of human ovarian cancer. In this model, a peritoneal human ovarian adenocarcinoma is induced by intraperitoneal injection of SKOV3 human ovarian tumor cells, into nude mice.
a) Naked siRNA Injection
The results show that siXL1 increases considerably the mice survival (60% survival at 130 days in the group treated with siXL1 versus 10% in untreated mice), whereas cisplatin treatment does not improve survival (
In the group treated with siXL1, the mice either die in the same interval as the control mice (50% of mice) or are cured, as demonstrated by the autopsy of mice after 150 days, showing no residual tumors in 50% of mice. The first half of the curves is identical in the different groups, indicating that the siRNA effect could be restricted to the smallest tumor nodules, maybe due to a direct effect of siXLl on endothelial cells of the blood vessels. By contrast, mice having larger tumors at the time of the siXL1 injection are less sensitive to siXL1 treatment in these conditions (naked siRNA administration) and their survival rate is similar to that of the untreated group. An heterogeneity in tumor development between mice, in this model could thus explained these results since there is one month delay between the death of the first and the last mouse in the untreated group.
Molecular effect of siRNA on its target was shown by the immunohistochemical analysis of Bcl-XL protein in SKOV3 peritoneal tumour nodes. Untreated mice tumours show high Bcl-XL expression, despite a relative intra and inter individual variability. Mice treated with various amounts of naked siXL1 (5, 25 or 100 μg/ml), reveal a relative intra and inter individual variability with some nodes showing a high Bcl-XL expression level while other nodes show a reduced Bcl-XL expression level. Nevertheless, the labelling intensity decreases as the siXL1 dose increases, suggesting a dose effect of the siRNA.
b) siRNA/PEI Complexes Injection
Immunohistochemical analysis of Bcl-XL protein in SKOV3 peritoneal tumour nodes, in mice treated with siXL1, naked or complexed with L-PEI, shows that the overall Bcl-XL protein expression level is lower in mice treated with siXL1/L-PEI complexes, compared to mice treated with naked siXL1. These results indicate that siXL1 delivery by a vector (L-PEI: linear polyethylenimine) could increase its activity in vivo.
This was confirmed by the subcutaneous tumor growth analysis, showing that siXL1 delivered with PEI and injected intraperitoneally, inhibits tumor growth for at least 45 days, whereas naked siXL1 injected in the same conditions has almost no effect, as tumor growth is almost similar to that of the untreated mice (